Transgenic algae engineered for higher performance

ABSTRACT

The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser. No. 13/037,149, filed Feb. 28, 2011, which claims the benefit of U.S. Provisional Application No. 61/308,974, filed Feb. 28, 2010, which is hereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No. DE-AC52-06NA25396, awarded by the United States Department of Energy to Los Alamos National Security, LLC. The government has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 87485-921786-SEQLIST.TXT, created on Mar. 2, 2015, 1430,105 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

FIELD

The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. The disclosure also relates to recombinant polynucleotides for the generation of transgenic algae having increased growth characteristics.

BACKGROUND OF THE INVENTION

The global biodiesel market demand is estimated to reach 37 billion gallons by 2016, growing at an average annual growth rate of 42%. Europe will be the major market for the next decade or so, closely followed by the US market. To meet this increased market demand, additional oil sources, especially non-edible oils, need to be explored (Li et al., 2008 Appl. Microbiol. Biotechnol. 80:749-756). Microalgae seems to be the only source of renewable biodiesel that has the potential to displace petroleum-derived transportation fuels without the controversial argument “Food or Fuel” and to help the nation reach the 2003 Biofuels Directive target of achieving greenhouse gas savings (Christi, 2007, Biotechnol. Adv. 25:294-306; Christi, 2008, Trends Biotechnol. 26:126-131; Cockerill and Martin, 2008, Biotechnol. Biofuels 1:9).

The most advanced biotechnology being applied to algal growth has been the creation of the antennae mutants that have less light harvesting machinery in the cell, which allows a greater fraction of the light to pass through an individual cell. This light then strikes other cells deeper in the culture. This is viewed as advantageous because some of the light energy striking a normal cell is in excess and is lost as fluorescence. These mutants do not suffer this loss of excess energy; it is available to other deeper cells in the culture. Thus the overall culture accumulates biomass faster. These mutants then grow using their normal rates of metabolism. In addition, some are attempting to engineer herbicide resistance genes into the production strains to allow competing algae in a production bioreactor to be controlled with the herbicide.

Numerous algal biofuels companies populate the landscape; it is reasonable to expect at least 20 of them will be producing algal oil at large scale within a year. Microalgal biodiesel is technically feasible (Gouveia et al. 2009 J. Ind. Microbiol. Biotechnol 36:269-274). However technoeconomic analyses show that for microalgal biofuels to be economically competitive with petrodiesel, the production, harvesting and extraction steps must be optimized and costs reduced. The production step must be increased substantially to increase the overall total biomass production. The degree to which the production rate can be improved within the constraints of the fixed costs of the production reactor, will dictate how much other costs must be reduced to achieve profitability or even the bottom line. The technology described herein can be expected to address that need.

In plants, the organic compound 2-oxoglutaramate is a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism. A number of transaminase and hydrolyase enzymes known to be involved in the synthesis of 2-hydroxy-5-oxoproline in animals have been identified in animal liver and kidney tissues (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197:304). In algae, the biochemical synthesis of 2-hydroxy-5-oxoproline has not been established. Moreover, the function of 2-hydroxy-5-oxoproline in algae is unknown.

Unkefer et al., U.S. Pat. No. 6,593,275, disclose a dramatic increase in the growth rate of algae when treated with 2-hydroxy-5-oxoproline. Continuously culturing the algae in the presence of this compound or mixtures of this compound with other prolines will enrich sub-strains of the algae that respond well to the prolines.

BRIEF SUMMARY OF THE INVENTION

The present disclosure relates to transgenic algae having increased growth characteristics. In one embodiment, the invention relates to transgenic algae having enhanced (faster) growth rates. Applicants have recently identified the enzyme glutamine phenylpyruvate transaminase (GPT) as a catalyst of 2-hydroxy-5-oxoproline (2-oxoglutaramate) synthesis in plants, and here disclose that transgenic algae engineered to over-express plant-derived GPT and glutamine synthetase (GS1) genes grow faster and produce higher amounts of chlorophyll compared to wild type algae.

In one embodiment, the disclosure provides the generation of GPT+GS1 Chlorella. The double-transgenic Chlorella demonstrated substantially faster growth rates that the untransformed Chlorella grown under identical conditions for the same amount of time. Methods for the generation of the transgenic algae of the invention are provided.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the algae is a green algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the algae is selected from a Chlorella or Chlamydomonas species.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GPT transgene is a plant-derived GPT.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is a green algae, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39, and wherein the GPT and GS transgenes are incorporated into the genome of the algae.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides the progeny of any generation of a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene and said GS transgene.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene and a GS transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 39, wherein the GPT and GS transgenes are incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to a wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) introducing a GS transgene into the algae or a progeny of the algae; (c) expressing the GPT transgene and the GS transgene in the algae or the progeny of the algae; and, (d) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not contain a GPT transgene or a GS transgene.

In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to a wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) introducing a GS transgene into the algae or a progeny of the algae; (c) expressing the GPT transgene and the GS transgene in the algae or the progeny of the algae; and, (d) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not contain a GPT transgene or a GS transgene, and wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the algae is a green algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the algae is selected from a Chlorella or Chlamydomonas species.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT transgene is a plant-derived GPT.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, and wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is a green algae, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, and wherein the GPT transgene is incorporated into the genome of the algae.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a progeny of any generation of a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT transgene is incorporated into the genome of the algae, and wherein said progeny comprises said GPT transgene.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is a green algae, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the algae is selected from a Chlorella or Chlamydomonas species, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT is an algal-derived GPT, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, wherein the GPT transgene is a plant-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID NO:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 47, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a transgenic algae including a GPT transgene, wherein said GPT transgene is operably linked to a promoter, and wherein the GPT is an algal-derived GPT, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO: 48, wherein the GPT transgene is incorporated into the genome of the algae, and wherein the transgenic algae displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed algae.

In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to an wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) expressing the GPT transgene in the algae or the progeny of the algae; and, (c) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not comprise a GPT transgene.

In one embodiment, the disclosure provides a method for increasing growth characteristics of an algae relative to an wild type or progenitor algae of the same species, the method including: (a) introducing a GPT transgene into the algae; (b) expressing the GPT transgene in the algae or the progeny of the algae; and, (c) selecting an algae having an increased growth characteristic relative to an algae of the same species that does not comprise a GPT transgene, wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Photograph of tissue culture plates, showing GPT+GS1 transformed Chlorella vulgaris on the right, and untransformed Chlorella vulgaris (control) on the left. See, Example 1, I nfra.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof (“polynucleotides”) in either single- or double-stranded form. Unless specifically limited, the term “polynucleotide” encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985 J. Biol. Chem. 260:2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “promoter” refers to a nucleic acid control sequence or sequences that direct transcription of an operably linked nucleic acid. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

The term “algae” refers to photosynthetic organisms of multiple phylogenetic groups, and includes numerous unicellular and multicellular species. The term “algae” as used herein includes organisms of the following phylogenetic groups: Chlorophyta (green algae; includes mostly fresh water species); Phaeophyta (brown algae; includes mostly marine species); Rhodophyta (red algae; includes mostly marine species); Chrysophyta; Xanthophyta; Bacillariophyta; Euglenophyta; Cryptophyta; Pyrrophyta; Raphidophyta; Haptophyta; Eustigmatophyta; Prasinophyta; Glaucophyta and Cyanobacteria (prokaryotic, blue-green algae). The class of algae which can be used in the methods of the invention is generally as broad as the class of algae amenable to transformation techniques.

Algae of the present disclosure include but are not limited to organisms of the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanidioschyzon, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Kappaphycus Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Laminaria, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Ostreococcus, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodactylum Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyra, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symbiodinium, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.

The terms “GPT polynucleotide” and “GPT nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a polypeptide involved in catalyzing the synthesis of 2-oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term “GPT coding sequence” refers to the part of the gene which is transcribed and encodes a GPT protein. The term “targeting sequence” refers to the amino terminal part of a protein which directs the protein into a subcellular compartment of a cell, such as a chloroplast. GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PCR products derived therefrom.

A “GPT transgene” is a nucleic acid molecule including a GPT polynucleotide which is exogenous to a transgenic algae harboring the nucleic acid molecule, or which is exogenous to an ancestor algae, of a transgenic algae harboring the GPT polynucleotide. More particularly, the exogenous GPT transgene will be heterogeneous with any GPT polynucleotide sequence present in wild-type algae into which the GPT transgene is inserted. To this extent, the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.

The terms “GS polynucleotide” and “GS nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term “GS coding sequence” refers to the part of the gene which is transcribed and encodes a GS protein. The terms “GS1 polynucleotide” and “GS1 nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase isoform 1 protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term “GS1 coding sequence” refers to the part of the gene which is transcribed and encodes a GS1 protein.

A “GS transgene” is a nucleic acid molecule including a GS polynucleotide which is exogenous to a transgenic algae, or which is exogenous to an ancestor algae of a transgenic algae harboring the GS polynucleotide. A “GS1 transgene” is a nucleic acid molecule including a GS1 polynucleotide which is exogenous to a transgenic algae harboring the nucleic acid molecule, or which is exogenous to an ancestor algae of a transgenic algae harboring the GS1 polynucleotide. More particularly, the exogenous GS or GS1 transgene will be heterogeneous with any GS or GS1 polynucleotide sequence present in wild-type algae into which the GS or GS1 transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.

Exemplary GPT from algae, higher plants and a fish are presented herein, and include GPT sequences derived from Chlorella, Arabidopsis, rice, barley, bamboo, soybean, grape, and zebra fish. GS and GS1 proteins are known; exemplary GS1 sequences provided herein include Arabidopsis and Hordeum.

Partial length GPT polynucleotides include polynucleotide sequences encoding N- or C-terminal truncations of GPT, mature GPT (without targeting sequence) as well as sequences encoding domains of GPT. Exemplary GPT polynucleotides encoding N-terminal truncations of GPT include Arabidopsis-30, -45 and -56 constructs, in which coding sequences for the first 30, 45, and 56, respectively, amino acids of the full length GPT structure of SEQ ID NO: 2 are eliminated.

In employing the GPT polynucleotides of the invention in the generation of transformed algal cells and transgenic algae, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only “substantially identical” to a sequence of the gene from which it was derived, as further defined below. The term “GPT polynucleotide” specifically encompasses such substantially identical variants. Similarly, one of skill will recognize that because of codon degeneracy, a number of polynucleotide sequences will encode the same polypeptide, and all such polynucleotide sequences are meant to be included in the term GPT polynucleotide. In addition, the term specifically includes those sequences substantially identical (determined as described below) with a GPT polynucleotide sequence disclosed herein and that encode polypeptides that are either mutants of wild type GPT polypeptides or retain the function of the GPT polypeptide (e.g., resulting from conservative substitutions of amino acids in a GPT polypeptide). The term “GPT polynucleotide” therefore also includes such substantially identical variants.

The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part 1: The Conformation of Biological Macromolecules (1980). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. “Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

The term “isolated” refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term “isolated” is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu. An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.

When percentage of sequence identity is used in reference to polypeptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Narl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nue. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, highly stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30° C. below the Tm. Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

Genomic DNA or cDNA including GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide sequences disclosed here. For this purpose, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37° C., and at least one wash in 0.2.times.SSC at a temperature of at least about 50° C., usually about 55° C. to about 60° C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.

Transgenic Algae:

The invention provides novel transgenic algae exhibiting faster growth and increased chlorophyll production. The transgenic algae of the invention are generated by introducing into algae cells one or more expressible genetic constructs capable of driving the expression of one or more polynucleotides encoding glutamine phenylpyruvate transaminase (GPT), and in some embodiments, one or more polynucleotides encoding glutamine synthetase (GS) and GPT.

The transgenic algae of the invention may be of any species capable of transformation, including those from the subphyla Chlorophyta, Chrysophyta, Phaeophyta, Rhodophyta, as well as the Cyanobacteria. In the last few years, successful genetic transformation of ˜25 algal species has been demonstrated, mostly via nuclear transformation (Hallmann, 2007, Transgenic Plant Journal 1:81-98). Among these, at least ten green algae species have been successfully transformed (mostly all unicellular species). Several species of red algae, brown algae and diatom species has also been reported (Hallmann, 2007, supra).

Improving the production step of algal biofuels production is being approached in more or less the standard ways of improving biological production, i.e., by optimizing nutrients, growth conditions and light/energy supply. Mineral nutrient supplies are being optimized through detailed studies of the need for each nutrient, and at what stoichiometry relative to other nutrients. The availability of carbon dioxide is being maximized typically with various delivery systems, each designed to maximize the amount of carbon dioxide dissolved in the water. As well, pH is being maintained at or near the optimum for growth.

Some attempts have been made to grow algal faster and more economically by establishing heterotrophic growth conditions. This has been achieved by providing fixed carbon, such as sugars, for the algae to use as carbon and energy sources. Algae also require an optimal amount of light to provide the energy for growth. The light impinging upon an algal cell is converted to chemical energy and used to drive the algal metabolism and cell growth. The amount of light striking algal cells in growing cultures is impacted by culture density, the distance the light must penetrate into the culture (i.e., the cells at the surface receive more light than cells further from the surface), and the amount of light receptors within an algal cell. Algal bioreactor designs are being tested that range from deep or shallow horizontal ponds to vertical glass/plastic reactors.

Alga cells over-expressing GPT and GS transgenes can be expected to take much better advantage of the optimized nutrients and the high availability of carbon dioxide, because they can be expected to increase their carbon dioxide fixing machinery, the ribulose bisphosphate carboxylase (RUBISCO) enzyme protein and activity state. If algae respond in the same way GPT+GS1 transgenic plants do (see co-owned, co-pending U.S. patent application Ser. No. 12/551,271), the over-expression of the GPT and GS transgenes in algae may be accompanied by increased expression of genes encoding RUBISCO subunits and the RUBISCO activase enzyme which controls the activity state of RUBISCO. The transgenic algae of the invention may also increase their capacity for and rate of uptake of nitrogen-based nutrients such as nitrate and ammonia. Such increased carbon fixation will result in increased flux through central metabolism and the incumbent increase in the concentration of organic acids that are known to induce production of the nitrogen uptake transporters.

In stable transformation embodiments of the invention, one or more copies of the expressible genetic construct become integrated into the host algae genome, thereby providing increased GS and/or GPT enzyme capacity into the algae, which may serve to mediate increased synthesis of 2-oxoglutaramate therein, which in turn signals metabolic gene expression, resulting in increased algal growth. 2-oxoglutaramate is a metabolite which is an extremely potent effector of gene expression, metabolism and plant growth (U.S. Pat. No. 6,555,500), and which may play a pivotal role in the coordination of the carbon and nitrogen metabolism systems in plants (Lancien et al., 2000, Enzyme Redundancy and the Importance of 2-Oxoglutarate in Higher Plants Ammonium Assimilation, Plant Physiol. 123:817-824).

The invention also provides methods of generating transgenic algae having faster growth rates. In one embodiment, a method of generating a transgenic algae having a faster growth rate, introducing into an algal cell an expression cassette including a nucleic acid molecule encoding a GPT transgene, under the control of a suitable promoter capable of driving the expression of the transgene, so as to yield a transformed algal cell, and obtaining a transgenic algae which expresses the encoded GPT. In another embodiment, a method of generating a transgenic algae having a faster growth rate comprises introducing into an algal cell one or more nucleic acid constructs or expression cassettes including nucleic acid molecules encoding a GPT transgene and an GS transgene, under the control of one or more suitable promoters (and, optionally, other regulatory elements) capable of driving the expression of the transgenes, so as to yield an algal cell transformed thereby, and obtaining a transgenic algae which expresses the GPT and GS transgenes.

GPT and GS transgenes suitable for use in generating the transgenic algae of the invention are described in co-owned, co-pending U.S. patent application Ser. No. 12/551,271. Other GPT polynucleotides suitable for use as GPT transgenes in the practice of the invention may be obtained by various means, as will be appreciated by one skilled in the art, tested for the ability to direct the expression of a GPT with GPT activity in a recombinant expression, or in a transient in planta expression system (U.S. Ser. No. 12/551,271, supra), or preferably in a transgenic algae.

Transgene Constructs/Algal Expression Vectors

In order to generate the transgenic algae of the invention, the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct), which can direct the expression of the transgene sequence in transformed algal cells. Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into an algal cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors. Once introduced into the transformed algal cell, the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a transient or stable fashion. Stable expression is preferred, and is achieved by utilizing transformation vectors which are able to direct the chromosomal integration of the transgene construct.

The basic elements of a nucleic acid construct for use in generating the transgenic algae of the invention are: a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed algae cell, the transgene(s) (i.e., GPT coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and sometimes other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).

Various plant, algae and animal GPT protein sequences and encoding DNA and GPT transgene expression constructs are presented in the Table of Sequences, infra. Similarly, various GS1 protein sequences and encoding DNA and GS1 transgene expression constructs are provided. These sequences are provided as examples and should not be considered limiting.

Typically, algae is transformed by causing the temporal permeabilization of the cell membrane, there by enabling vector DNA to enter the algal cell. DNA integration occurs naturally, by recombination events, resulting in ectopic integration into the algal genome, resulting in stable events. Biolistic approaches, such as particle bombardment, are typically used to transform algal cells. Polyethylene glycol mixtures using DNA coated particle has also been successfully utilized. Additionally, certain wall-reduced algae strains have been employed, in order to achieve protoplast transformation in the presence of polyethylene glycol and the transgene construct. Electroporation and even Agrobacterium-mediated transformation of algae has been reported. For details, and references to published reports of various transformation protocols and vector systems, see Hallmann, 2007, supra for review.

Any promoter capable of being functional in algae may be used to direct the expression of the GPT or GPT+GS transgene constructs. In preferred embodiments, the promoter may be an endogenous algal promoter. Various vectors used to transform algae are known, including plasmid vectors which become integrated into the nucleus of algal cells and there direct the cytoplasmic expression of the transgene products (i.e., plasmid pSSCR7, Davies et al., 1994, Plant Cell 6:53-63). Other nuclear-directed vectors direct transgene expression products to the periplasm. One such vector is a derivative of pSSCR7, modified to incorporate a 5′ aryl sulfatase periplasmic targeting transit sequence (Davies et al., 1994, supra). Still other vectors direct transgene integration to the chloroplast plastome by homologous recombination, whereby transgene expression is localized to the chloroplast (Hutchinson, et al., 1996, In: Molecular Genetics of Photosynthesis, Frontiers in Molecular Biology. Anderson B., Salter A H, and Barber J. eds., Oxford University Press, pp. 180-196).

EXAMPLES

Various aspects of the invention are further described and illustrated by way of the example which follows, which is not intended to limit the scope of the invention.

Example 1 Generation of Transgenic Chlorella Materials and Methods:

Chlorella vulgaris strain #259 was purchased from Culture Collection of Algae at the University of Texas (UTEX, Austin, Tex.), and maintained as recommended on Bristol medium.

Two transgene expression vectors were assembled and used to transform the algae with electroporation. The first vector was the Cambia 1201 vector containing Tomato Rubisco small subunit promoter and Arabidopsis GS (SEQ ID NO: 45). The second vector was the Cambia 1305.1 vector containing 35S CMV promoter Zea mays full length GPT gene sequence [SEQ ID NO: 46]

Electrotransformation was used to insert these vectors simultaneously into the Chlorella culture; this was carried out according to the method of K. C. Chow and W. L. Tung. (Electrotransformation of Chlorella vulgaris. Plant Cell Reports 1999 18:778-780). The antibiotic selection was Hygromycin B at a concentration of 20 micrograms/ml. Bristol peptone digest media (UTEX web site) was used to culture the cells on plates.

Results:

Transformed and control algae were plated out identically. Twelve days later, 15 dark green colonies were observed growing on the transformed plate and none on the control plate. FIG. 1 shows a photograph of the transformed (right) and untransformed (control, left) Chlorella, taken 23 days post-plating. Vigorous green cultures were seen only on the transformed algae plate. In addition, the transformed algae colonies showed far greener and darker coloration compared the control algae. The dark green is chlorophyll, and the increase in chlorophyll per cell has been observed as a characteristic of the faster growing plants. Finally, the colony count at day 15 post-plating, and the much greater colony numbers at 23 days post-plating, are consistent with faster growth.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. 

1. A transgenic alga comprising a plant-derived or algal-derived glutamine phenylpyruvate transaminase (GPT) transgene and a glutamine synthetase transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a promoter.
 2. The transgenic alga according to claim 1, wherein the alga is a green alga.
 3. The transgenic alga according to claim 2, wherein the green alga is a Chlorella species.
 4. The transgenic alga according to claim 1, wherein the GPT transgene is a plant-derived GPT.
 5. The transgenic alga according to claim 1, wherein the GPT is an algal-derived GPT.
 6. The transgenic alga of claim 4, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 44, and SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID N0:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO:
 47. 7. The transgenic alga according to claim 5, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO:
 48. 8. The transgenic alga according to claim 1, wherein the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 39, an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, an amino acid sequence that is at least 75% identical to SEQ ID NO: 7, and an amino acid sequence that is at least 75% identical to SEQ ID NO:
 39. 9. The transgenic alga according to claim 1, wherein the GPT and GS transgenes are incorporated into the genome of the alga.
 10. A progeny of any generation of the transgenic alga of claim 9, wherein said progeny comprises said GPT transgene and said GS transgene.
 11. The transgenic alga of claim 9, which displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to a control wild-type or untransformed alga.
 12. A method for increasing growth characteristics of an alga relative to an wild type or progenitor alga of the same species, comprising: (a) introducing a plant-derived or algal-derived GPT transgene into the alga; (b) introducing a GS trans gene into the alga or a progeny of the alga; (c) expressing the GPT transgene and the GS transgene in the alga or the progeny of the alga; and, (d) selecting an alga having an increased growth characteristic relative to an alga of the same species that does not comprise the GPT transgene or the GS transgene.
 13. The method according to claim 12, wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions.
 14. A transgenic alga comprising a plant-derived or algal-derived GPT transgene, wherein said GPT transgene is operably linked to a promoter.
 15. The transgenic alga according to claim 14, wherein the alga is a green algae.
 16. The transgenic alga according to claim 15, wherein the green alga is a Chlorella species.
 17. The transgenic alga according to claim 14, wherein the GPT transgene is a plant-derived GPT.
 18. The transgenic alga according to claim 14, wherein the GPT is an algal-derived GPT.
 19. The transgenic alga of claim 17, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 44, and SEQ ID NO: 47, an amino acid sequence that is at least 75% identical to SEQ ID NO: 2, an amino acid sequence that is at least 75% identical to SEQ ID NO: 9, an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, an amino acid sequence that is at least 75% identical to SEQ ID NO: 19, an amino acid sequence that is at least 75% identical to SEQ ID NO: 21, an amino acid sequence that is at least 75% identical to SEQ ID NO 24, an amino acid sequence that is at least 75% identical to SEQ ID NO: 30, an amino acid sequence that is at least 75% identical to SEQ ID N0:31, an amino acid sequence that is at least 75% identical to SEQ ID NO: 32, an amino acid sequence that is at least 75% identical to SEQ ID NO: 33, an amino acid sequence that is at least 75% identical to SEQ ID NO: 34, an amino acid sequence that is at least 75% identical to SEQ ID NO: 35, an amino acid sequence that is at least 75% identical to SEQ ID NO: 36, an amino acid sequence that is at least 75% identical to SEQ ID NO: 44, and an amino acid sequence that is at least 75% identical to SEQ ID NO:
 47. 20. The transgenic alga according to claim 18, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, and an amino acid sequence that is at least 75% identical to SEQ ID NO:
 48. 21. The transgenic alga according to claim 14, wherein the GPT transgene is incorporated into the genome of the alga.
 22. A progeny of any generation of the transgenic alga of claim 21, wherein said progeny comprises said GPT transgene.
 23. The transgenic alga of claim 21, which displays a faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions when compared to a control wild-type or untransformed alga.
 24. A method for increasing growth characteristics of an alga relative to an wild type or progenitor alga of the same species, comprising: (a) introducing a plant-derived or algal-derived GPT transgene into the alga; (b) expressing the GPT transgene in the algae or the progeny of the alga; and, (c) selecting an alga having an increased growth characteristic relative to an alga of the same species that does not comprise the GPT transgene.
 25. The method according to claim 24, wherein the increased growth characteristic is selected from the group consisting of: faster growth rate, increased chlorophyll production, increased ribulose bisphosphate carboxylase (RUBISCO) level, increased nitrogen use efficiency, increased biomass yield, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, and/or increased tolerance to salt or saline conditions. 